The objectives are to determine the mechanisms by which preconditioning protect from ischemic injury. Preconditioning refers to a period of resistance to ischemic injury, that can be induced by temporary ischemic, hypoxia or adenosine A1 receptors, prior to a period of sustained ischemic. Neither at the natural trigger, the target tissue (nerve, vascular, myocyte or interstitial), nor the mechanisms responsible for in situ preconditioning have been established. To date, preconditioning has only been shown to occur in intact hearts or animals. A new observation is that isolated adult rabbit cardiomyocytes can be endogenously preconditioned against subsequent ischemia, by a brief, substrate-free, oxygenated incubation period. This preconditioning conferes a high degree of protection (1-2 hour delay in death) from subsequent ischemia. In vitro- preconditioning mechanisms do not appear related to energy utilization/production, or to catacholamines. This proposal, focuses on characterization of this new model, as a basis for future studies of endogenous cellular mechanisms of preconditioning. The aims are to determine; A. the relations between protection and high energy phosphate levels; B. if the trigger, and protection are/are not analogous to that in intact hearts; C. if preconditioning decays during a post incubation period prior to ischemia; D. determine if the pathways of preconditioning, and protein phosphatase inhibitors converge to a common PKC-related pathway; E. determine if preconditioning can be induced in vitro, in another species. This new in vitro model will be useful in determination of mechanisms of endogenous cellular myocardial protection, which could allow development of a new class of therapeutic agents to reduce infarct size, or lead to better methods of organ preservation.